The role of cortactin-mediated actin branch stabilization in force-producing actin networks

PROJECT SUMMARY
Networks of branched actin filaments produce forces necessary for cellular processes ranging from cell
migration to endocytosis. These branches are formed by proteins actin-related protein 2/3 (Arp2/3) complex,
along with proteins that promote Arp2/3 activation. Cortactin is a multi-functional protein that can activate
Arp2/3 complex on its own at high concentrations, synergize with certain nucleation promoting factors, or
stabilize branches after they are formed. This multifunctionality has made isolating the role of cortactin as a
branch stabilizer difficult. Actin branches are intrinsically stable, so why cortactin-mediated branch stabilization
is important for regulating force-producing actin networks is unknown. One possibility is that cortactin protects
branches from being removed by a class of debranching proteins, such as coronin 1B or glia maturation factor-
γ. Additionally, actin networks are age-segregated, with newer branches near the surface being thought to be
more important in the ability of the network to provide pushing forces, so the importance of cortactin may be
related to whether debranchers target the younger or older regions of the network. In this proposal, we will
utilize a reconstituted assay to isolate cortactin's role as a branch stabilizer, and determine whether it is
important for force production by preventing branches from being removed prematurely in Aim 1. Neural
Wiskott-Aldritch syndrome protein will be used as a nucleation promoting factor, because our lab has
previously shown does not synergize with cortactin. Lower concentrations of cortactin will minimize cortactin-
mediated branch nucleation. Bead motility assays will allow for observing force production by age-segregated
actin networks that push against beads in the presence of cortactin alone and with differentially targeted
debranching proteins. In doing so, we will determine how the interplay between branch stabilization and
destabilization affects the ability of actin networks to provide pushing forces. Many debranchers have also
been implicated in bundling actin, so cryo-electron tomography will identify whether actin branches dissociate
or remodel as bundles. Then, in Aim 2, we will assess how these proteins affect the recycling of monomeric
actin from branched actin networks. By incorporating the filamentous actin disassembly protein, cofilin, into the
bead motility assay, we will assess whether the interplay between cortactin and debranching proteins on
sustaining bead motility over longer periods of time, which will be verified by an actin co-precipitation assay
and cryo-electron tomography. Finally, in Aim 3, we will characterize the activity of a more enigmatic
debrancher, coronin 7. In addition to the bead motility assay to identify the role of coronin 7 on force production
by branched actin networks, we will utilize total internal reflection fluorescence microscopy to observe the
frequency of debranching in the presence of coronin 7 with or without cortactin. We will then observe binding
and dissociation of fluorescently labeled coronin 7 to actin filaments made from ATP (newer branches) or ADP
(older branches) to assess nucleotide preference.

Grant Number: 5F32GM156029-02
NIH Institute/Center: NIH
Principal Investigator: Broderick Bills